The use of optically detectable labeling groups, and particularly those groups having high quantum yields, e.g., fluorescent, phosphorescent, luminescent or chemiluminescent groups, is ubiquitous throughout the fields of analytical chemistry, biochemistry, and biology. In particular, by providing a highly visible signal associated with a given reaction, one can better monitor that reaction as well as any potential effectors of that reaction. Such analyses are the basic tools of life science research in genomics, diagnostics, pharmaceutical research, and related fields.
For example, fluorescence-based optical assays utilizing fluorescent dye labels are frequently used in scientific analyses. The fluorescence detected in a fluorescence-based optical assay is the result of a three-stage process that occurs in the fluorophores or fluorescent dyes present in a reaction mixture. The first stage is excitation in which a photon with quantized energy from an external light source having a specific wavelength (e.g., from a laser) is supplied and absorbed by a fluorophore creating an excited electronic singlet state (S1′). The second stage is the excited-state lifetime in which the excited fluorophore undergoes several different changes to relax its energy to the lowest singlet state (S1). From the S1 state several possible mechanisms can occur in the third stage, fluorescence, in which a photon of energy (S1-S0) is emitted returning the fluorophore to its ground state. Many thousands of these three-stage processes of excitation and emission typically occur to produce a signal detectable by standard optical sensors.
One of the many pathways that dissipate the energy of the excited electronic singlet state is the intersystem crossing (ISC), involving a change in spin multiplicity, transiting the electron from S1 to the excited triplet state (T1). In many fluorescent dye molecules the formation of the much longer life-time triplet-state species greatly reduced the brightness of the fluorescence emission. In addition, it exhibits a high degree of chemical reactivity in this state, which often results in photobleaching and the production of damaging free radicals.
Analyses using optically detectable labeling groups have generally been performed under conditions where the amounts of reactants are present far in excess of what is required for the reaction in question. The result of this excess is to provide ample detectability, as well as to compensate for any damage caused by the detection system and allow for signal detection with minimal impact on the reactants. For example, analyses based on fluorescent labeling groups generally require the use of an excitation radiation source directed at the reaction mixture to excite the fluorescent labeling group, which is then separately detectable. However, one drawback to the use of optically detectable labeling groups is that prolonged exposure of chemical and biochemical reactants to such light sources, alone, or when in the presence of other components, e.g., the fluorescent groups, can damage such reactants, e.g., proteins, enzymes, and the like. The traditional solution to this drawback is to have the reactants present so far in excess that the number of undamaged reactant molecules far outnumbers the damaged reactant molecules, thus minimizing or negating the effects of the photo-induced damage.
A variety of analytical techniques currently being explored deviate from the traditional techniques. In particular, many reactions are based on increasingly smaller amounts of reagents, e.g., in microfluidic or nanofluidic reaction vessels or channels, or in “single molecule” analyses. These analytic systems provide for the observation of only one or a few “events” at a time. For example, such events could be the binding of an antigen to an antibody, a ligand binding to a receptor, cleavage of a polymer (e.g., nucleic acid, protein, or saccharide polymer), incorporation of a unit into a polymer (e.g., an amino acid into a protein, a nucleotide into a nucleic acid, etc.). Such low reactant volumes are increasingly important in many high throughput applications, such they can provide data that is not attainable when observing a plurality of molecules in the more traditional ensemble approaches.
One challenge in performing single-molecule (or few-molecule) reactions comprising labeled reactants is being able to distinguish a labeled reactant engaged in an event under observation from other labeled reactants that are free in the reaction mixture. This is especially important for intermolecular events that require high concentrations of reactants, e.g., to ensure adequate binding to an enzyme catalyzing the reaction. As such, the labeled reactants in the reaction mixture can emit “background” noise that obscures detection of a signal from the event of interest in the reaction mixture. In such analyses, the term “signal-to-noise ratio” refers to a measure that compares the level of a desired signal (“signal”) to the level of background signal, or “noise.”
Another challenge in performing reactions based upon increasingly smaller amounts of reagents is that such reactions are more severely impacted by photo-induced damage (e.g., photobleaching and free radical formation). For example, photo-induced damage of the enzyme component in a single molecule reaction can completely stop the reaction and prevent further data acquisition.
As such, the present disclosure is directed, inter alia, to methods and compositions that result in (i) an increased signal-to-noise ratio (SNR) in a reaction mixture; (ii) increased photoprotection, or both during illuminated reactions. Increases in SNR can facilitate the detection of reactants when they are participating in an event under observation, e.g., at a reaction site, and thus provide useful improvements to the methods and compositions currently available. For example, methods and compositions that increase the signal-to-noise ratio would not only enhance detection of signals of interest, but could also allow higher concentrations of reactants in various analytical systems. Increased photoprotection in illuminated reactions can enhance the detection of signals of interest and allow reactions to progress for longer periods of time or under more intense illumination conditions, thereby increasing data acquisition and/or allowing higher intensity illumination signals to be employed. These improvements in SNR and photoprotection can lead to increased accuracy in signal detection assays, e.g., single-molecule sequencing reactions.